Synapses are the connections between nerves and form the basis of neural activities in animals. As such, defects in synapse formation have been implicated in many neurological disorders of humans. The mechanisms leading to the formation of synapses remain poorly understood and controversial despite the identification of many of the central players involved in the process. It remains unclear how these players work in vivo to form functional synapses. In this proposal, zebrafish will be used to address this question. The zebrafish in vivo system is unique in the experimental merits it offers, including the ease of optical studies, tools for genetic manipulation, and the availability of viable mutants with defects in synaptic proteins. Nicotinic acetylcholine receptors (AChR) and rapsyn will be the focus of this proposal. AChR receives direct input from the nerve and brings about muscle contraction, while rapsyn is intimately involved in the clustering of AChRs at the synapse. Zebrafish mutant lines have been identified for both AChR and rapsyn. The null line of AChR, caused by the lack of its delta subunit, affords the opportunity to express modified forms of AChRs to identify its functional domains, which is not possible with any other animal model system. Also, a potential tyrosine phosphorylation site that mediates AChR synaptic clustering has been identified. This AChR-null line, in combination with the rapsyn mutant, will be used to determine the AChR-rapsyn interactions leading to synaptic clustering, with a particular emphasis on the role of tyrosine phosphorylation in this process. In the proposed experiments, synaptic molecules are expressed with fluorescent tags, and their expression is traced in living animals. The use of mutants and the expression of modified forms of synaptic molecules will clarify the roles played by these molecules and indicate how they work as a whole to form functional synapses. These experiments will provide an understanding of the synapse in general, leading to better therapeutic strategies for human neurological disorders.